Assays in which a sample and one or more reagents are variously reacted to form a ligand/conjugate complex such as an antibody/antigen or similar complex, which may then be observed in order to measure the presence or level of a predetermined moiety in the sample, are well known. Typically, an antibody is used to assay for the presence of an antigen for which the antibody is specific. These assays have been extended to quantitate haptens such as hormones, alkaloids, steroids, antigens, antibodies, nucleic acids, and fragments thereof, enzymes, and cell surface receptors. It is in this broad sense that the term “ligand/conjugate” as used herein should be understood.
Sensitive immunoassays typically use tracer techniques in which a tagged constituent of the complex is incorporated, for example in the reagent, the non-complexed tagged reagent then being separated from the complexed reagent. Thereafter, the complex can be quantitated by observing a signal from the tag. Radioisotopes, fluorescent and chemiluminescent molecules, calorimetric tags, and other markers have been used to label constituents or moieties of the complex, appropriate apparatus being employed to detect and measure the radiation from the label.
In such assays where at least one component of the complex is initially bound to a solid substrate preparatory to formation of the complex, a basic problem arises because of the typically lengthy time required to bind that component to the solid substrate such as a well microtiter plate or bead, sometimes requiring incubation times on the order of hours for binding of a component to the solid phase to occur notwithstanding such expedients as heating, agitation and the like. Consequently, there is a significant amount of prior art regarding attempts to reduce this incubation time, including using microbeads, dipsticks, macrobeads, etc., but nonetheless incubation times on the order of 10 to 20 minutes are typical.
There are numerous formats for solid phase assays, but they can nonetheless be sorted into two types: sandwich and competition, both of which are well known to those skilled in the art. Sandwich assays typically require the antigen to be able to simultaneously bind to more than one conjugate. One of the conjugates is attached to the solid phase while the other conjugate is labeled with a tag. The amount of tagged conjugate attached to the solid phase is then related to the antigen concentration in a sample. A universal problem in sandwich assays is nonspecific binding, i.e., the amount of labeled conjugate that is on the solid phase, but not attached to the antigen. In designing sandwich assays, there is usually a trade-off between signal level and nonspecific binding. Increasing the concentration of labeled conjugate or incubation time of the labeled conjugate will increase the signal levels, but will also increase the amount of nonspecific binding.
Various approaches have been used to try to reduce the effect of nonspecific binding including wash buffers, detergents, blocking steps, referencing, etc., which are also well known in the art.
In competition assays a labeled moiety, either a conjugate to or an analog of the antigen, can be bound to the solid phase. The presence of antigen reduces or inhibits the binding of the labeled moiety to the solid phase. The amount of inhibition in the signal is a measure of the antigen concentration. For a competition assay to work well the amount of antigen, labeled moiety and solid phase binding sites must be roughly equal. Therefore, competition assays are usually much less sensitive than sandwich assays and also suffer from a small linear range. They are nonetheless useful for small antigens which can only bind with one conjugate at a time.
The results of chemical, biochemical, and biological assays are used to make important decisions, and therefore, the accuracy and reliability of the result is of utmost importance. Heretofore, control samples of known concentration are assayed periodically, or even simultaneously with the sample to be measured, to calibrate and verify the operation of the assay on the unknown sample. This process reduces, but does not eliminate, the possibility of error in the assay of interest.
An object of the present invention is to provide a solid phase assay method which solves many of the problems described above. Another object of the invention is to provide a “competition-like” assay with an increased linear range of determination, improved sensitivity, reduced susceptibility to errors caused by deteriorating reagents or variations in environmental conditions, reduced susceptibility to errors caused by bubbles or other mechanical problems, ease of automation, and reduced time to obtain a final result of the assay, among other things, relative to standard competition assays known in the art. A “competition-like” assay of the invention is similar to a standard competition assay except that at least one step of the competition-like assay of the invention is time limited in a manner described herein so that competitive equilibria typical of standard competition assays are not established.
Another object of the invention is to provide an improved sandwich assay in which at least one step is time limited so that problems that are associated with non-specific binding reactions and are typical of standard sandwich assays are avoided.
Yet another object of the invention is to eliminate time-consuming incubation steps in a solid phase assay method or to significantly reduce the required time to run the assay.